The repair of DNA damage is crucial to survival of all organisms. Thus, it is rfot surprising that the major DMA damage repair pathways, such as nucleotide excision repair and mismatch repair, are conserved from bacteria to man. These pathways are efficient and, for the most part, do not require the chromosomal DNA replication machinery for their activity. How do cells deal with the encounter between a replication fork and template DNA damage? What happens when the damage itself inactivates the replication fork?, creating a requirement for both replication fork restart and repair of the damage. As a result of studies from a number of groups, many centered, as ours have been, on the properties of PriA and its gene, a new paradigm has emerged describing the replication of the bacterial chromosome. This paradigm holds that even under normal growth conditions, the replication forks formed at oriC become inactivated as a result of an encounter with endogenous DNA template damage. This creates a requirement for both repair of the damage and reactivation of the replication forks. Our studies in the previous grant period have demonstrated that the <)>X174-type primosome is required for replication fork reactivation where it directs the assembly of a new replication fork on DNA substrates that are generated by the action of the recombination proteins. Furthermore, genetic data suggests that there are multiple pathways of replication fork restart involving different combinations of the primosomal proteins. In order to understand completely this intersection of two of the major pathways of DNA metabolism, we will model replication fork demise and reactivation in vitro. We will proceed by asking the following questions: What is the fate of the enzymatic components of the replication fork after a collision with either template DNA damage or a frozen protein-DNA complex? How is replication fork demise affected by the location and type of damage to the DNA? What are the DNA structures left at stalled replication forks? What conditions lead to DNA breakage at stalled replication forks? What is the biochemical basis for the existence of multiple pathways of replication fork restart? And, does the manner of recombination protein-directed processing of the DNA at a stalled fork direct the enzymatic pathway of replication forkreactivation? Using purified recombination and replication proteins, we will study the demise and reactivation of replication forks formed in isolated replisome complexes at oriC on small plasmid minichromosomes that have been engineered to carry specific types of DNA damage in specified locations.